The major goal of the studies proposed in this continuation grant is to elucidate ore fully the physiological and biochemical strategies employed by renal medullary/ papillary epithelial cells (nephron segments) which permit them to function in their osmotically harsh environment in vivo. The cells of these nephron segments must possess both the ability to return cell volume to some nominal value after osmotic shrinkage in hypertonic media (Volume Regulatory Increase, VKI) and after osmotic swelling in hypotonic media (Volume Regulatory Decrease, VRD), and to maintain cell enzyme (metabolic) function during exposure to near molar interstitial salt and urea concentrations. Osmotic adaptation by both plant and animal cells depends on altering total cell solute content by complex processes involving: (1) uptake or efflux of inorganic ions via transport mechanisms located in plasma membranes; and/or (2) cellular synthesis or uptake of organic solutes, including polyols, amino acids and methylamines. Some evidence indicates that these special organic "osmolytes" may protect cell enzymes from hypertonic osmotic stress. To investigate these complex osmoregulatory issues (types of osmolyte solutes, mechanisms of uptake or synthesis, controlling factors, effects on metabolism and enzyme function) in renal medullary cells, the proposed research program will incorporate the talents of several investigators/collaborators to provide a broad integrative investigative approach. Three cell systems derived from rat and mouse will be utilized for these studies, including in vitro perfusion of nephron segments, suspensions of nephron segments and cells, and primary cell cultures. Optical, electrophysiologic, tracer, fluorescence, biochemical, electron probe and nuclear magnetic resonance methodologies will be used with these cell systems to test the following hypotheses: (i) that all medullary nephron segments are capable of rapid VRI and VRD responses and that these processes are mediated by cellular fluxes of inorganic ions; (ii) that VRI requires antidiuretic hormone; (iii) that VRI is a two-phase process involving a rapid uptake of inorganic ions (primarily NaCl) and a slower synthesis and/or accumulation of organic solutes; and (iv) that cellular accumulation of organic osmolytes is vital to maintenance of normal cellular function during hypertonicity. The results of these studies will provide not only basic information on how renal medullary/papillary cells adapt to osmotic stress, but also, insights into how other mammalian cells, including those in the brain, adjust to altered body fluid tonicity.